Project Summary Solid tumors contain not only cancer cells, but also non-malignant cells such as fibroblasts, endothelial cells, and macrophages. In recently years, multiple lines of evidence have made it clear that these non- malignant cells, which together comprise the ?tumor microenvironment?, play crucial roles in disease progression and therapeutic response. Specifically, the activation of key signaling pathways in non-malignant cells of the microenvironment can render nearby tumor cells resistant to anticancer drugs. However, our understanding of the landscape of pathways that govern microenvironment-driven resistance is highly limited by the fact that no methods exist to perform large-scale pharmacological or genetic screening of the microenvironment. To address this challenge, we recently developed a microfluidics-based platform that enables the parallel co-culture of non-malignant and malignant cells in thousands of miniature, on-chip ?apartments?. In this proposal, we describe a strategy of systematically activating key signaling pathways in non-malignant cells using pooled lentiviral cDNA libraries, then assessing the impacts of these perturbations on drug sensitivity in nearby breast cancer cells. Specifically, we will use this strategy to map the microenvironmental signaling pathways that control the sensitivity of breast cancer cells to approved and emerging targeted therapies, then credential and validate hits from these assays using an integrated experimental and computational pipeline. Thus, this project will yield the first screening platform for large-scale functional dissection of cell-cell interactions and define key, clinically relevant microenvironmental pathways that shape anticancer drug responses.